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Fractionated TMT32 bridge channel samples from healthy PBMC donor for generation of FAIMS RTLS spectral libraries

ABSTRACT: This is the first of three MassIVE repositories associated with the manuscript "Single-Cell Proteomics of Human Peripheral Blood Mononuclear Cells Exceeding 600 Cells per Day." This repository contains datasets generated from a healthy human PBMC donor to create a spectral library for RTLS analysis. Bulk PBMC peptides were TMTpro 32-plex labeled (channels 126 and 135ND, pooled 1:1), microfractionated, and analyzed using FAIMS-enabled LC-MS/MS (Orbitrap and ion trap MS2) on a ThermoFisher Orbitrap Eclipse Tribrid mass spectrometer. [PBMC Isolation, Digestion, and Cleanup]: Cryopreserved PBMCs were thawed, washed, and lysed in 8 M urea (50 mM TEAB). Proteins were quantified by BCA, diluted, and sequentially digested with Lys-C and trypsin (1:50 enzyme-to-protein ratios). Peptides were desalted using C18 MacroSpin columns, concentrated, quantified, and dried prior to labeling. [TMTpro Labeling]: Peptides were reconstituted in 50 mM bicine (pH 8.5). Two 10 ug aliquots were labeled with TMTpro channels 126 or 135ND, quenched with hydroxylamine, pooled, and diluted to 0.2 ug/uL. [High-pH Microfractionation]: TMT-labeled peptides were separated by high-resolution reverse-phase capillary LC (nanoACQUITY UPLC) using a 200 um x 65 cm C18 column. Fractions were collected every minute from 65-170 min, concatenated in a staggered pattern, dried, and reconstituted in 2% acetonitrile/0.1% formic acid. [LC-MS/MS Analysis]: Samples were analyzed using a homebuilt dual-column nanoPOTS autosampler coupled to an Orbitrap Eclipse Tribrid MS with FAIMS. Columns were operated at 100 nL/min with a 75-min gradient. FAIMS compensation voltages of -45 and -65 V were used. Two spectral library acquisition methods were applied: 1) Orbitrap MS2: HCD fragmentation, 120k MS1 resolution, Orbitrap MS2 detection; 2) Ion Trap MS2: HCD fragmentation with rapid ion trap MS2 detection. [Database Searching and Spectral Library Generation]: Raw files were processed in FragPipe v23.0 using MSFragger, MSBooster, Percolator, IonQuant, and TMT-Integrator. Searches used the UniProt Homo sapiens database (FASTA 08/16/24) with 20 ppm precursor tolerance, tryptic/semi-tryptic specificity, static TMTpro modifications on lysine and N-termini, and methionine oxidation as a variable modification. Protein FDR was set to 1%.MS2 spectra were filtered to the top 150 fragment ions and converted to mzML. Spectral libraries were generated in SpectraST for each FAIMS CV and MS2 analyzer, converted to mzVault .db format, and merged to produce final Orbitrap and ion trap spectral libraries for -45 V and -65 V FAIMS conditions.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: James M. Fulcher Ljiljana Pasa-Tolic

PROVIDER: MSV000100682 | MassIVE | Sat Jan 31 16:26:00 GMT 2026

SECONDARY ACCESSION(S): PXD073879

REPOSITORIES: MassIVE

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Project description:This is the second of three MassIVE repositories associated with the manuscript "Single-Cell Proteomics of Human Peripheral Blood Mononuclear Cells Exceeding 600 Cells per Day." This repository contains TMTpro 32-plex-labeled datasets from a healthy human PBMC donor, including both bulk (10 ng) and single-cell proteomic measurements. [PBMC Isolation, Digestion, and Cleanup]: Cryopreserved PBMCs were thawed, washed, and lysed in 8 M urea (50 mM TEAB). Protein concentration was measured by BCA, followed by dilution and sequential digestion with Lys-C and trypsin (1:50 enzyme-to-protein ratios). Peptides were desalted using C18 MacroSpin columns, concentrated, quantified, and dried prior to labeling. [TMTpro Labeling of Bulk Bridge Sample]: Bulk PBMC peptides were reconstituted in 50 mM bicine (pH 8.5). Two 10 ug aliquots were labeled with TMTpro channels 126 and 135ND, quenched with hydroxylamine, pooled, and diluted to 0.2 ug/uL for use as a bridge sample. [Single-Cell Isolation (cellenONE)]: PBMCs were thawed, washed, and resuspended in PBS prior to single-cell isolation using a cellenONE instrument. Cells were selected based on size, circularity, and elongation to exclude debris, apoptotic cells, and doublets. Individual cells were dispensed into 30 wells of 32-plex N2 nanoPOTS chips, with two wells reserved for bridge sample addition. Chips were stored at -80 deg C until processing. [N2 nanoPOTS Single-Cell Sample Preparation]: Single cells were digested in nanoliter volumes using TMT-compatible buffer containing Lys-C and trypsin and incubated overnight at 37 deg C. TMTpro 32-plex reagents were dispensed directly onto each well using the cellenONE. Labeling proceeded overnight, followed by quenching with hydroxylamine, acidification with formic acid, evaporation, and storage at -20 deg C. TMTpro-labeled bridge sample was added at levels described in the manuscript. [LC-MS/MS Analysis]: Samples were analyzed using a homebuilt dual-column nanoPOTS autosampler coupled to a ThermoFisher Orbitrap Eclipse Tribrid mass spectrometer with FAIMS. Dual parallel columns enabled near-continuous MS operation. Peptides were separated at 100 nL/min using a 75-min gradient. FAIMS compensation voltages of -45 and -65 V were applied. Two acquisition strategies were used: 1) Standard MS2 Method: Orbitrap MS2 detection following HCD fragmentation; 2) RTLS Method: Initial ion-trap MS2 scans were searched in real time against FAIMS-specific spectral libraries, triggering Orbitrap MS2 acquisition for matched precursors. Library matching used a 20 ppm precursor tolerance and cosine score >= 40. [Database Searching and Quantification]: Raw files were processed using FragPipe v23.0 with MSFragger, MSBooster, Percolator, Philosopher, IonQuant, and TMT-Integrator. Searches used the UniProt Homo sapiens database (FASTA 08/16/24). For RTLS datasets, only high-resolution Orbitrap scans were retained for database searching. Search parameters included 20 ppm precursor tolerance, semi-tryptic specificity, static TMTpro modification on lysine and peptide N-termini, and methionine oxidation as a variable modification. Protein-level FDR was set to 1%. TMT quantification was performed using IonQuant and TMT-Integrator with median aggregation and MS1-based ratio-to-abundance conversion.

Project description:This is the third of three MassIVE repositories associated with the manuscript "Single-Cell Proteomics of Human Peripheral Blood Mononuclear Cells Exceeding 600 Cells per Day." This repository contains 2,130 single PBMC measurements (71 raw files) acquired using a dual-column LC system with real-time library searching (RTLS). [PBMC Isolation, Digestion, and Cleanup]: Cryopreserved PBMCs from a healthy donor were thawed, washed, and lysed in 8 M urea (50 mM TEAB). Protein concentration was determined by BCA, followed by dilution and sequential digestion with Lys-C and trypsin (1:50 enzyme-to-protein ratios). Peptides were desalted using C18 MacroSpin columns, concentrated, quantified, and dried prior to labeling. [TMTpro Labeling of Bulk Bridge Sample]: Bulk PBMC peptides were reconstituted in 50 mM bicine (pH 8.5). Two 10 ug aliquots were labeled with TMTpro channels 126 and 135ND, quenched with hydroxylamine, pooled, and diluted to 0.2 ug/uL for use as a bridge sample. [Single-Cell Isolation (cellenONE)]: PBMCs were thawed, washed, and resuspended in PBS before single-cell isolation using a cellenONE instrument. Cells were selected based on size, circularity, and elongation to exclude debris, apoptotic cells, and doublets. Individual cells were dispensed into 30 wells of 32-plex N2 nanoPOTS chips, with two wells reserved for bridge sample addition. Chips were stored at -80 deg C until processing. [N2 nanoPOTS Single-Cell Sample Preparation]: Single cells were digested overnight at 37 deg C in nanoliter volumes using TMT-compatible buffer containing Lys-C and trypsin. TMTpro 32-plex reagents were dispensed directly into each well using the cellenONE. Labeling proceeded overnight, followed by quenching with hydroxylamine, acidification with formic acid, evaporation, and storage at -20 deg C. TMTpro-labeled bridge sample was added at levels described in the manuscript. [LC-MS/MS Analysis with RTLS]: Samples were analyzed using a homebuilt dual-column nanoPOTS autosampler coupled to a ThermoFisher Orbitrap Eclipse Tribrid mass spectrometer with FAIMS. Dual parallel columns enabled near-continuous MS operation. Peptides were separated at 100 nL/min using a 75-min gradient. FAIMS compensation voltages of -45 and -65 V were applied. RTLS was implemented using rapid ion-trap MS2 scans searched in real time against FAIMS-specific spectral libraries. Precursors passing a cosine similarity threshold >= 40 triggered high-resolution Orbitrap MS2 acquisition. Orbitrap MS1 scans were acquired at 120,000 resolution, followed by HCD fragmentation and Orbitrap MS2 detection for RTLS-selected precursors. [Database Searching and Quantification]: All raw files were processed using FragPipe v23.0 with MSFragger, MSBooster, Percolator, Philosopher, IonQuant, and TMT-Integrator. Searches used the UniProt Homo sapiens database (FASTA 08/16/24). Only high-resolution Orbitrap scans were retained for database searching. Search parameters included 20 ppm precursor tolerance, semi-tryptic specificity, static TMTpro modification on lysine and peptide N-termini, and methionine oxidation as a variable modification. Protein-level FDR was set to 1%. TMTpro quantification was performed using IonQuant and TMT-Integrator with median aggregation and MS1-based ratio-to-abundance conversion.

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Fractionated TMT32 bridge channel samples from healthy PBMC donor for generation of FAIMS RTLS spectral libraries

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